1. Field of the Invention
The present invention relates generally to the fields of endocrinology and neurobiology. More particularly it concerns the mechanisms by which extracellular Ca.sup.2+ and the steroid hormone 1,25 (OH).sub.2 vitamin D.sub.3 modulate vascular smooth muscle force generation.
2. Description of Related Art
Essential hypertension is a major health problem in the U.S. with an estimated 50 million adults being affected (Gifford, 1993) and is characterized by an increase in peripheral resistance in the face of normal cardiac output (Folkow, 1982). There is a clear pattern of inheritance and influence of the environment (Lifton, 1996) and untreated hypertension is a significant risk factor for stroke, myocardial infarction, coronary artery disease, renal failure, and premature death (Gordon et al., 1977). Standard therapy is directed toward lowering blood volume (diuretics and salt restriction), or reduction of vascular tone (vasodilators, pressor antagonists, and sympatholytics) (The Fifth Report of the Joint National Committee on Detection, Evaluation, and Treatment of High Blood Pressure, 1993). Although medical management of hypertension has contributed to a large reduction in stroke incidence over the past two decades, reduction in risk of myocardial infarction has not shown a parallel improvement (Anderson et al., 1991). Thus, cardiovascular disease remains the number one cause of death in the U.S.
Available antihypertensive compounds have unwanted side effects or clear contraindications. Diuretics and beta adrenoreceptor antagonists are indicated as first line therapy (The Fifth Report of the Joint National Committee on Detection, Evaluation, and Treatment of High Blood Pressure, 1993) but are associated with significant untoward effects including erectile dysfunction and fatigue (.beta.-blockers), and are contraindicated in renal failure (diuretics). These observations underscore the need for novel therapeutic approaches to hypertensive disease. One area of blood pressure research which has potential for the development of novel pharmacological strategies, but has remained untapped, is the study of the relationship between Ca.sup.2+ homeostasis and blood pressure regulation.
Relationship Between Systemic Ca.sup.2+ Homeostasis and Blood Pressure.
Evidence linking blood pressure and systemic Ca.sup.2+ homeostasis includes the clinical observation that primary hyperparathyroidism is associated with hypertension and that parathyroidectomy frequently lowers blood pressure (Mallette et al., 1974; Massry et al., 1986), epidemiologic data associating regional hardness of water with death from cardiovascular disease (Sharrett and Feinleib, 1975), and Ayachi's seminal demonstration that elevated dietary calcium intake lowers blood pressure in the spontaneously hypertensive rat (SHR) (Ayachi, 1979) which has been amply confirmed (Arvola et al., 1993; Bukoski and McCarron, 1986; DiPette et al., 1989; Kageyama and Bravo, 1987; McCarron et al., 1981). Epidemiologic surveys (Ackley et al., 1983; Belizan and Villar, 1980; Fogh-Anderson et al., 1984; Garcia-Palmieri et al., 1984; Harlan et al., 1984; McCarron et al., 1982) and clinical trails (Bucher et al., 1996a; 1996b; Grobbee and Hofman, 1986; McCarron and Morris, 1985; Strazullo et al., 1986) also support a link between Ca.sup.2+ intake and blood pressure in humans. For example, analysis of NHANES data showed a significant inverse correlation between blood pressure and calcium intake (McCarron and Morris, 1982); and surveys of pregnant women showed a correlation between gestational hypertension and calcium intake (Belizan and Villar, 1980; Bucher et al., 1996a). Although clinical trials have shown only a small overall blood pressure lowering effect of Ca.sup.2+ supplementation (McCarron and Morris, 1985), sub-groups have been shown to respond to Ca.sup.2+ supplementation with a significant fall in blood pressure (Resnick et al., 1985a; 1985b).
Mechanisms Linking Ca.sup.2+ Homeostasis with Blood Pressure.
Several hypotheses have been proposed to explain the apparent link between Ca.sup.2+ homeostasis and blood pressure and have been recently reviewed (Bukoski et al., 1995). These include Ca.sup.2+ induced modulation of calciotropic hormone levels, alterations in Na and water balance, or changes in sympathetic nerve activity. In contrast with these ideas, work in two areas has prompted proposal of a novel hypothesis which states that the perivascular CaR, acting as a sensor for extracellular Ca.sup.2+, responds to changes in interstitial Ca.sup.2+ concentration with the release of a local vasodilator substance. One area was the discovery that extracellular Ca.sup.2+ relaxes isolated arteries at low physiologic concentrations (Bian et al., 1995); the other was the molecular demonstration of the parathyroid CaR (Brown et al., 1993b; Garrett et al., 1995c).
Modulation of Vascular Tone by Extracellular Ca.sup.2+.
It has long been recognized that extracellular Ca.sup.2+ can suppress arterial force generation (Bohr, 1963; Cow, 1911; Holman, 1958). The physiologic significance of this action of Ca.sup.2+ has been unclear, however, since only very high concentrations of extracellular Ca.sup.2+ have generally been shown to induce relaxation (Bohr, 1963; Hollaway and Bohr, 1973; Webb and Bohr, 1978). Recent work, however, has demonstrated that raising extracellular Ca.sup.2+ from as little as 1.0 mM to 1.5 mM relaxes isolated arteries (Bian et al., 1995) and that cumulatively raising extracellular Ca.sup.2+ above 1.5 mM causes nearly complete relaxation with an ED.sub.50 value of 2.4.+-.0.17 mM, n=12. Relaxation induced by Ca.sup.2+ dependent on the release of an endothelium-derived relaxing factor, or on the production of NO, but is associated with the release of a vasodilator substance from the adventitial surface of the artery. These results, and observations that Ca.sup.2+ induced relaxation is associated with decreased myofilament Ca.sup.2+ sensitivity and can be blocked by K.sup.+ channel antagonists led to the proposal that a Ca.sup.2+ receptor that is similar or identical to that described in parathyroid gland plays a mediating role in contrast to persistent reports that the CaR is not expressed in vascular smooth muscle (Brown et al., 1993a; 1995).
Ca.sup.2+ Homeostasis and Cell Function.
Serum ionized Ca.sup.2+ is normally regulated within tight limits and is a function of the amount of Ca.sup.2+ absorbed by the intestine, reabsorbed from the load filtered by the kidney, and the net sum of Ca.sup.2+ deposition into and resorption from the bone mass. A simplified view of the endocrine mechanisms that regulate Ca.sup.2+ homeostasis can be gained by considering the dynamic systemic responses that occur in response to changes in serum Ca.sup.2+ (Bukoski et al., 1995). An increase in serum ionized Ca.sup.2+ is recognized by the membrane spanning, G protein coupled CaR of the parathyroid cell which in turn elicits a decrease in the release of parathyroid hormone (PTH) (Brown et al., 1993a; 1995). The rise in Ca.sup.2+ also activates a CaR on the thyroid C cell which increases calcitonin release (Garrett et al., 1 995a,b). A fall in serum Ca.sup.2+ has the opposite effect of increasing PTH and decreasing calcitonin. PTH acts at the level of bone to increase release of Ca.sup.2+ into the plasma, and the fall in calcitonin releases its suppressive effect on bone Ca.sup.2+ resorption. PTH also acts as the kidney where it increases reabsorption of Ca.sup.2+ and stimulates the production of 1,25 (OH).sub.2 vitamin D.sub.3 which in turn acts via genomic and non-genomic mechanisms to increase intestinal absorption and renal reabsorption of Ca.sup.2+ (Nemere et al., 1993). The net result is an increase in serum Ca.sup.2+ at the expense of changes in calciotropic hormone levels.
Two important concepts for the working hypothesis are implicit in this model. One is that interfaces exist in the epithelial linings of the gut and kidney, and adjacent to metabolically active bone cells where gradients in interstitial Ca.sup.2+ can be generated. The gradients expose adjacent tissues to levels of interstitial Ca.sup.2+ that are significantly different from that which is present in the mixed venous plasma (e.g. serum ionized Ca.sup.2+). This elevation in interstitial Ca.sup.2+ may significantly modulate local cell function. The second concept is that changes in Ca.sup.2+ ion content in the mixed plasma are of sufficient magnitude to stimulate Ca.sup.2+ receptors on the parathyroid and thyroid C cells and to elicit changes in second messengers affecting PTH and calcitonin release. The CaR is therefore exquisitely sensitive to small, i.e., approximately 10th millimolar, changes in extracellular ionized Ca.sup.2+.
The Ca.sup.2+ Receptor (CaR).
It has recently been shown that parathyroid cells express a cell surface "Ca.sup.2+ receptor" (CaR) which enables them to detect and respond to small changes in the concentration of extracellular Ca.sup.2+ (Brown et al., 1993b). The parathyroid CaR is a G protein coupled receptor possessing a large extracellular domain and showing homology only with the metabotropic glutamate receptors. The parathyroid CaR is composed of 1078 amino acids in the human and 1079 in the rat and the native receptor is heavily glycosylated in both species. To date, only one gene for the CaR has been identified although there is some evidence for alternatively spliced forms and deletion/insertion mutations (Pollack et al., 1993). In an effort to explain high sensitivity Ca.sup.2+ -induced relaxation, it has been proposed that the CaR gene product may be present in the arterial wall (Bian et aL, 1995; Bukoski et al., 1995).
The discovery and molecular characterization of the Ca.sup.2+ receptor was the culmination of studies of the cellular mechanisms by which extracellular Ca.sup.2+ regulates the function of specific cell types, particularly the parathyroid cell. A breakthrough in this area was the cloning of cDNA encoding the CaR from the bovine parathyroid (Brown et al., 1993a). The coding region of the full length CaR cDNA is 3,237 bp and predicts a 120 kDa protein. This protein has 7 putative membrane spanning domains, intracellular protein kinase C phosphorylation sites, and a large extracellular domain with multiple glycosylation sites. The CaR has now been cloned from the human parathyroid (Garrett et al., 1995a,b), rat and human medullary thyroid carcinoma cell (Garrett et al., 1995a,b), rat kidney (Riccardi et al., 1995), and brain (Ruat et aL., 1995). Previously, attempts at localizing the CaR in the vascular wall, using northern blot analysis of aortic tissue, had failed to show the appropriate transcript.
Among the important pharmacologic properties of the CaR is the fact that it can be activated by polyvalent cationic molecules including the trivalent cations La.sup.3+ and Gd.sup.3+, neomycin, and spermine (Brown, 1991). This property led to the development of a novel class of phenylalkyamine derivatives by Nemeth and colleagues (1986; 1987; 1995). These compounds are the only potent and selective agonists that have been reported and are presently used in clinical trials for the treatment of hyperparathyroidism (Fox et al., 1993; Nemeth et al., 1995).
Recently is has been demonstrated that physiological levels of extracellular Ca.sup.2+ significantly modulate force generation by resistance arteries (Bian et al., 1995a; 1995b). This observation, when coupled with independent observations that Ca.sup.2+ in the interstitial space of tissues that are involved in transcellular Ca.sup.2+ movement, i.e., the intestine, kidney, and bone, may be significantly greater than that which is present in the mixed venous plasma (Brown, 1991), provides a strong argument for the physiological significance of modulation of vascular tone by extracellular Ca.sup.2+.
Modulation of Smooth Muscle Function by Extracellular Ca.sup.2+.
It is well established that smooth muscle contraction is triggered by a rise in intracellular Ca.sup.2+ which activates actomyosin ATPase through a myosin light chain phosphorylation dependent mechanism (Stull et aL, 1991). Although still controversial, it is also clear that steady state force maintenance is a Ca.sup.2+ dependent process that depends upon either the formation of the latch bridge state (Hai et al., 1989) or a thin filament regulated mechanism (Zhang, et al., 1994).
Since a rise in intracellular Ca.sup.2+ is critical for smooth muscle contraction, it is often assumed that extracellular Ca.sup.2+ serves only as a storage reservoir for the transmembrane Ca.sup.2+ gradient. Evidence that this is an oversimplification dates back to 1911 when Cow studied different mammalian arteries and showed that slightly supraphysiologic concentrations of Ca.sup.2+ depress force generation (Cow, 1911). Approximately 50 years later, Bohr (1963) showed that Ca.sup.2+ has a dual effect on smooth muscle; high concentrations of extracellular Ca.sup.2+ suppress the early force response of the aorta to epinephrine, while the later steady state response is enhanced. This inhibitory effect of extracellular Ca.sup.2+ was attributed to a "membrane stabilizing" effect and has been proposed to result from binding of Ca.sup.2+ to the cell membrane surface which in turn decreases lipid bilayer mobility transmembrane Ca.sup.2+ via transport proteins and channels (Dominiczak et al., 1990, Dominiczak et al., 1991).
Further characterization of the phenomenon of Ca.sup.2+ induced relaxation showed that raising Ca.sup.2+ from 4.1 to 20.1 mmol/L relaxes rat tail arteries (Webb et al., 1978); and the effect is antagonized by pre-treatment with ouabain, low Na, and low temperature. It was concluded that Ca.sup.2+ induces relaxation by activating the Na pump. Wu and colleagues (1978) later showed that K.sup.+ -induced contraction of rat aorta is depressed as extracellular Ca.sup.2+ from 0 to 5.1 mM caused a graded contraction in cremaster arterioles of the rat (Joshua et al., 1988).